The bla TEM-1 b-lactamase gene has become widespread due to the selective pressure of b-lactam use and its stable maintenance on transferable DNA elements. In contrast, bla SME-1 is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of bla SME-1 via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, bla SME-1 was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 b-lactamase. Taken together, these results suggest that fitness costs associated with some b-lactamases may limit their dissemination.